Normal human diploid fibroblasts have a limited doubling potential in tissue culture. This is to be contrasted with malignant or transformed mammalian cells which appear to have unlimited doubling potential. The cause of such difference is not fully understood Polyamines (putrescine, spermidine and spermine) are ubiquitous biogenic organic cations, the rate-controlling enzyme for the biosynthesis of polyamines in mammalian cells is ornithine decarboxylase (ODC). Both ODC and polyamines have been shown to be intimately related to growth regulation. High ODC and polyamine contents are generally associated with proliferative growth. It is proposed that alteration of polyamine metabolism during the aging process may lead to the permanent loss of doubling potential of human diploid cells. To test this hypothesis, we will investigate the following parameters in normal human diploid fibroblasts as a function of their cellular population doubling level (PDL): (i) the regulation of ODC; (ii) the intracellular content of polyamines and their metabolites as determined by High Performance Liquid Chromatography (HPLC); (iii) the uptake and excretion of polyamines; (iv) the metabolic labeling of an 18,000-dalton protein by (14C)putrescine, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography; (v) the metabolic conversion of (14C)putrescine to (14C)amino acids via (14C)alpha-aminobutyric acid, and the incorporation of (14C)amino acids into cellular proteins; (vi) the biodegradative enzymes of polyamines. In addition to the normal human diploid fibroblasts, genetically defective fibroblasts derived from patients with premature aging disease will also be used for comparison. The proposed studies should yield information on the roles of polyamines in the aging process. The data generated from these studies will also enable us to better understand the biological functions of polyamines and ODC in cell growth regulation. The long range goal of this research include (a) to understand the basic mechanism of the cellular aging process, and (b) to search for parameter(s) which may serve as a biochemical index of the age of a cell population and thus can be used to design an assay to determine the age of both primary cell cultures and established cell strains.